Molecular techniques, in particular analyses of nucleic acids, gain more and more importance in the field of diagnostics. In particular, the molecular diagnostics in the field of respiratory diseases, such as pneumonia or tuberculosis, often require the analysis of bodily samples such as body fluids which are very diverse in their appearance, e.g., blood, sputum, and tracheal secretions. To date, each sample type requires a different lysis buffer, processing method, and lysis procedure which are often not transferable to other sample types. No protocol has been published so far which covers all sample types relevant for molecular diagnostics of respiratory diseases, e.g., pneumonia. Thus, it is currently not possible to use a standardized, user-friendly routine protocol for all sample types relevant for diagnosis of pneumonia patients. Thus, lysis buffers and processing methods which are universally applicable to a wide variety of bodily samples, in particular, samples which are relevant for the diagnosis of respiratory diseases, such as pneumonia, are desired.
It is particularly important for patients with severe acute infections of the respiratory tract to rapidly identify causative pathogens and concomitant risk factors, such as drug resistances, to enable a switch from the initial broad-band antibiotic therapy to a customized therapy specifically targeting causative pathogens with their identified drug resistances. International guidelines for severe pneumonia subtypes, such as hospital-acquired, ventilator-acquired, or healthcare-associated pneumonia, strongly recommend identification of certain risk pathogens (e.g., Pseudomonas aeruginosa) or risk factors for multi-drug resistances (e.g., mecA) which have a significant impact on the design of the antibiotic regimen.
The diagnostic approaches used to date for respiratory diseases, e.g., chest radiography, smear microscopy, physical examination, or microbial culture tests, e.g., of sputum cultures, cultures of bronchoscopic samples, or blood cultures, are often not suitable to identify pathogens or risk factors and/or have very low efficiencies. Thus, to date, there is no efficient detection method which is supported by validated clinical studies for the diagnosis of pneumonia. As a consequence, pneumonia guidelines do not recommend any diagnostic approach as a gold-standard method. Rather, a combination of different diagnostic methods covering different samples is used based on the expertise and experience of the attending physician. In this context, several respiratory as well as non-respiratory samples are used for the diagnosis. For example, blood cultures are used as indicators for bacteraemia or pleural punctates as indicators for empyema which leads to a high risk for mortality for pneumonia patients.
Samples which are relevant for the diagnosis of respiratory diseases are generally difficult to handle. Such samples encompass sputum, pus, pleural fluid, gastric aspirate, endotracheal aspirate, transtracheal aspirate, bronchoalveolar lavage, laryngeal swab, nasopharyngeal swabs, and others which are usually inhomogeneous mixtures of many different components of different chemical and physical behavior. Generally, such samples are highly viscous and even samples of the same type differ vastly in their composition. However, accessibility and lysis of inflammatory pathogens can be less efficient if they are trapped in a solid and viscous environment.
All diagnostic methods used so far aiming at detection of pathogens in samples of the respiratory tract require laborious sample processing for decontamination and liquefaction using a combination of enzymes such as proteases, lipases, DNases, or glycosidases, detergents, chaotropic agents, chelating agents, and reducing agents among others. However, some of these agents such as SDS are known inhibitors of nucleic acid amplification and analysis methods. Furthermore, due to the high infection risk any treatment of tuberculosis suspected samples requires an S3 environment with certified laminar flows and extensive protection measures to exclude any exposure of personnel to live bacteria. Thus, for molecular tests it would be of advantage to use bodily samples directly for nucleic acid diagnostics and circumvent the handling intensive decontamination and liquefaction procedures.
Thus, molecular diagnostics in respiratory diseases is currently time-consuming, laborious, error-prone, and associated with a high risk of contamination. Furthermore, since each sample type requires a different handling procedure, processing in a high-throughput setting is not possible.
Therefore, it is the aim of the present invention to provide a lysis buffer and a simple processing method which are universally applicable for the lysis of a variety of different sample types, such as bodily samples relevant for the diagnosis of respiratory diseases, and which reduce the need for intensive handling of the samples, and thus, reduce the risk of contamination and allow for molecular diagnostics in a high-throughput setting.
The present inventors have surprisingly found that a lysis buffer containing as the only active ingredients a chaotropic agent, a reducing agent, and a protease is universally applicable for the lysis of bodily samples, in particular, of bodily samples that are relevant for the diagnosis of respiratory diseases. Thus, the lysis buffer according to the present invention is universally applicable, for example, for the lysis of gastric juices and sputum or tracheal secretion samples, which are highly different in their compositions.
Furthermore, the present inventors have developed a processing method, which is easy and safe to perform, universally applicable, automatable, applicable in a high-throughput setting, and which does not require any laborious and time-consuming decontamination or liquefaction pre-treatments of the samples. In particular, the processing method of the present invention allows performing the complete lysis procedure of a bodily sample in a single reaction tube, without the need for centrifugation and sample transferring or pipetting steps, substantially reducing the required hands-on work and contamination risk. The lysate resulting from the processing method according to the present invention can be directly transferred to nucleic acid isolation procedures, for example, based on silica membranes, silica beads, or magnetic beads technology, without the need for further nucleic acid purification methods such as phenol-chloroform extraction or precipitation. Thus, the lysates are, for example, suitable for direct transfer to a QiaAmp™ DNA isolation column without significant clogging of the column, which was not expected, in particular, for the highly viscous samples of the respiratory tract.